The transfer of genetic material (DNA) from one species to another has been a focus of research for years. Transgenic animals are animals containing transferred exogenous genetic material which is passed on to their offspring. Once founder animals are established, they pass transgenic traits on to some, or all, of their offspring.
Manufacture of improved foods and agricultural products has been a focus of transgenic technology. One aim of transgenic technology is the production of useful recombinant proteins in milk or blood of farm animals.
Transgenic technology has also enabled in vivo study of gene expression. The in vivo results may often be directly related to a specific disease process, thus yielding a greater understanding of the disease process. Transgenic animals may also be used for the production of transplant organs which do not cause the usual immunogenic reactions in a recipient, e.g., a human recipient.
The potential for transferring genetic material into mammalian cells cultured in vitro has existed for many years. However, gene transfers into whole mammalian organisms have only recently been practicable.
Mosaic mice (i.e., non-transgenic mice having exogenous DNA in some of their tissue) have been produced by injection of tetracarcinoma cells into the blastocysts of developing mice (Brinster, R. L., J. Exp. Med., 140: 1049-1056 (1974); Mintz et al., Proc. Natl. Acad. Sci. U.S.A., 72: 3585-3589 (1975); and Papaioannou et al., Nature, 258: 69-73 (1975)). Teratocarcinoma cells have also been used as vehicles for introducing genes into mice to produce mosaic mice. (See, for example, Pellicer et al., Proc. Natl. Acad. Sci. U.S.A., 77: 2098-2102 (1980)).
Mosaic mice produced by the above methods have reduced germline transmission. This is due to the mosaic mice that develop as males being unable to produce sperm.
Liposome technology (anionic and cationic) has provided efficient genetic transformation of mammalian cells cultured in vitro. As a logical extension of in vitro technology, several researchers have attempted to generate transgenic animals via liposomes complexed with DNA. These attempts employed both conventional mammalian cell transfection techniques and microinjection into the cytoplasm and perivitelline space. No transgenic animals resulted. See, e.g., Loskutoff, et al., Theriogenology, 25, 169 (1986); or Reed, et al., Theriogenology, 25, 293 (1988).
Another advance in non-embryonic in vitro mammalian cell transfection technology uses normal cellular processes, such as receptor-mediated endocytosis, to incorporate DNA into mammalian cells. Endocytosis has enabled insertion of genetic material into an in vitro non-embryonic cellular genome. Wu et al., J. Biol. Chem., 263, 14621-14624 (1988) covalently linked a ligand for a specific cell-surface receptor to polylysine (a polycation which binds DNA by electrostatic interaction). The polylysine ligand-complex was allowed to bind DNA. Cells were then incubated with the polylysine-ligand/DNA complex, which resulted in the uptake and expression of the exogenous DNA. However, the exogenous DNA was not incorporated into the host cell's genome. The polylysine served as a bridge between the DNA and the ligand for a specific cell surface receptor. The cell surface receptor was a critical component, since it was required for endocytic absorption of the DNA.
The invention of embryonic stem cell technology has made it possible for the first time to transfer DNA into an intact mammal. Genetic transfers with stem cells have been severely limited because obtaining stem cells and making gene transfers is labor-intensive and costly. Stem cells must be cultured in vitro for long periods of time. The result is usually germline mosaic animals. Stem cell technology has the further limitation of only having been successfully demonstrated in mice. Consequently, this technology cannot yet be applied to the generation of farm animals where gene transfers would have important practical applications.
DNA has been inserted, by microinjection, directly into a non-germ cell being cultured in vitro, (Capecchi, M. Cell, 22: 479-488 (1980)). The technique was later extended to pronuclear microinjection of an early embryo to produce transgenic animals. (Wagner, et al. Proc. Natl. Acad. Sci. U.S.A. 78, 6376-6380 (1981)).
Pronuclear microinjection of DNA produces fertile transgenic animals, which may yield offspring having the transgenic trait. However, there are significant disadvantages with pronuclear microinjection.
An important negative side effect of pronuclear microinjection is a dramatic reduction in the embryonic viability of microinjected embryos. Pronuclear micro-injected embryos show a significant loss in embryonic viability as compared to uninjected embryos.
Brinster, et al., Proc. Natl. Acad. Sci. U.S.A. 82, 4438-4442 (1985) compared cytoplasmic injection of plain buffered DNA to pronuclear injection of the same material. While cytoplasmic injection was found less detrimental to in vitro embryo survival than pronuclear injection, it was not recommended for producing transgenic mice. Cells from only 2 embryos of the 224 fetuses examined tested positive for the foreign DNA. Only fetal cells were tested for the foreign DNA, and no transgenic live pups were produced by cytoplasmic injection. Importantly, the embryos were not tested for mosaic traits versus true transgenic traits.
In further cytoplasmic microinjection attempts, neither the Brinster, et al. authors or other researchers (e.g., King, et al., Mol. Reprod. Dev. 1, 57-62 (1988)) have been able to duplicate the above results. No exogenous DNA has been detected in animal cells or tissues, which have arisen from embryos injected with just the exogenous naked DNA in a buffer. This raises the possability that the result reported by Brinster, et al., Proc. Natl. Acad. Sci. U.S.A. 82, 4438-4442 (1985) is a false positive for transgenic DNA.
Accordingly, there is a need for a method which provides greater viability of embryos. Greater viability would be particularly important in producing transgenic farm animals such as sheep, goats and cows. Transgenic animals from these species are difficult to efficiently produce, since they only have a small number of offspring at a time (from 1 to 4). Further, there is a need for an improved method for producing transgenic species where pronuclei are difficult to see (e.g., sheep, goats, cows, fish, or birds). For those species, a method of gene transfer not requiring a visualized pronucleus would be desired.